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MicroTwJC: Lasering Bacteria !

This week on Microbiology Twitter Journal Club, we'll be seeing what happens when we hit bacteria with LASERS.

Picture is unrelated

Well, we aren't specifically targeting bacteria with Lasers, we are using them to "activate" a chemical.
The chemical in question is Methylene Blue. It was one of a number of different dye chemicals discovered towards the end of the 19th century that Paul Ehrlich attempted to use to treat bacterial diseases. The basic story is that Ehrlich, a microscopy afficianado, loved to dye bacteria to look at them under a microscope, and then realised that some dye compounds really loved to attach to the surface of bacteria. He decided that this property would be very useful for targeting drugs against microbes.
It had been used to treat Malaria, but stopped being used around the second world war, and isn't as effective as modern drugs. It is relatively safe to use, but has some really cool side effects (It turns the whites of your eyes blue and makes your pee blue as well) so it has also been used occasionally as a placebo.
But we aren't interested in this molecules natural antibiotic ability. We are interested what happens to it when we shine a Laser at it. This causes Methylene Blue to attack the nearest oxygen and break it apart to form Singlet Oxygen. Singlet Oxygen reacts with everything, they can break apart or mutate DNA strands, attack lipids and break down sugars, which makes it lethal for bacteria and for most living things*. It is very difficult for bacteria to form any kind of defence against this kind of attack. Any new enzymes or lipids they produce can be easily be broken apart by singlet oxygen.

Bacteria like Staphylococcus aureus can perform a neat trick to mitigate the effects of antibiotic therapy. Certain mutations slow down their growth, creating "Small Colony Variants". The slowing of the bacterial metabolism mitigates the effects of certain antibiotics that work by specifically attacking it. These Small Colony Variants (SCV) tend to emerge when an infection has been treated with antibiotics over a long period of time, and they tend to have acquired (or evolved) resistance genes**.
One property of these SCV's is that they don't bind to certain antimicrobial compounds as easily, which means that Methylene Blue may not be able to bind to it, which may limit its effectiveness.
So the question this paper wants to answer is whether these "Small Colony Variants" can be killed using Singlet oxygen generated by Laser-activated methylene blue.

The researchers obtained some SCV's of Staphylococcus aureus that were resistant to the antibiotic known as Kanamycin.
In Figure 1 they took an SCV strain (Grey bars) and the Parental strain (White bars) from which it evolved, and tested them for their sensitivity to Laser activated Methylene blue.
They had the bacteria suspended in a nutrient solution lit up by a Laser beam set at a constant intensity, and added different concentrations of Methylene Blue.

The bacteria only start to be killed off when the methylene blue reaches a concentration of 10 micromolar. They use an ANOVA to work out whether all the samples are statistically similar, and the stars indicate the samples that would not fit if we assumed the all the samples had the same mean. Unfortunately, this test did not allow them to compare the parent and wild type strains directly. This would require a Two-way ANOVA, or Post tests between individual columns. They could have done more experiments in between 5 micromolar and 20 micromolar and attempt to fit a regression line***.
Nevertheless, this data shows that SCVs are killed off by laser light only when Methylene Blue is concentrated at 10 micromolar.
The next step is to see what happens when we change the intensity of the laser light. We would want to use as low intensity of Laser light as possible, for reasons I will get into later.
The effects of changing Laser light intensity is shown in Figure 2.

They suspended the bacteria in 20 micromolar Methylene Blue and then shot more Laser light at it at increasing intensity. The greater the intensity of the Laser light, the greater the degree of bacterial killing.
Whilst the graph appears to clearly show the SCV's are more resistant to laser light than their non-SCV counterparts, we don't know this for certain because the statistics yet again don't compare them to each other. The one way ANOVA assumes all the data are part of the same distribution, and simply points out the data that don' fit in. It still proves their point, that laser light kills bacteria, but it really limits them.

I'll give a quick example. You see the columns at 1.93 J/cm^2 in this figure ? They should be really similar to the columns on the previous figure at 20uM. In fact, if you look at the axes, you will find that the data are very similar. This is because they were exposed to the same conditions. Yet one set of these columns has significance stars above it, and the other does not. Why is this ?

Imagine the average for each data set. Figure 1 has a lot of columns with high bacterial counts compared to Figure 2. Since ANOVA's work by comparing data to the assumed average for a whole population, the averages for the two data sets are going to be different, with Figure 2 having the lower average. This is why we have what is known as a Type 2 error in Figure 2. If the researchers used an analysis that could compare these datasets more effectively, or to better establish trends, then they could do so much more with their data than they could a the moment.

But even this limited application of the Statistics backs up the authors interpretation of the data. That pretty much sums up this study for me. The authors demonstrate exactly what they set out to do.

Wider Applications

The main reason for performing this research is for Photodynamic Therapy. The idea is that you would give a patient methylene blue, and then fire laser light at it to get rid of bacteria. Long time readers may note that I have had some reservations with photoactivation treatments in a previous MicroTwJC. All of those issues were relevant for deep tissue infections. However, in this case, the researchers are targeting infections occurring on the skin, or in exposed wounds, and my previous statements don't completely apply in this situation. For things like nasal decontamination, the bacterium is assumed to be on the surface of the skin, or adhered to the hairs of the nasopharynx. So this therapy should be okay.

Summary
All in all, it's not a bad paper. I may still reeling from the last MicroTwJC, because I'm happy that they at least tried to do some statistics, even if they did misapply them. I may be giving them the benefit of the doubt this time around, but I can give this paper a firm stamp of Okay.

EDIT:Points raised during #Microbiology Twitter Journal ClubBiofilms
I don't usually make edits to these sum ups unless I am absolutely convinced that I missed something big in my initial reading of the paper.
The first thing I missed out was pointed out by Jana Hiltner.

I understood that they wanted to use it for superficial infections, so I was thinking biofilm and was surprised they used 1/2 #microtwjc
— Jana Hiltner (@Janitensen) October 15, 2013

They didn't test it out on biofilms, which makes extrapolating efficacy from this data into an in vivo situation really difficult. But I realised it's worse than that. Even if they did utilise standard biofilm assays, it would still not tell us anything about whether this would make a useful nasal decolonisation procedure.

Most of the standard biofilm assays I am aware of submerse bacteria in a nutrient medium.
The humans are one of the few animals that possess a dry nose. Now, these differences would not be too problematic, save for one problem. The Methylene blue associated photoactivation of singlet oxygen only occurs when both compounds are dissolved in water. Water is a crucial intermediate in this reaction, and so the efficacy of this reaction would not be very good in the dry atmosphere of the nasopharynx.
So in light of this knowledge, I am going to have to retract what I said about this paper's conclusion's being "okay".

There were a couple of parts of this paper where the term "data not shown" turn up. The authors invoke this in two separate occasions. Let us examine both of those cases, and see the degree to which it justifies the criticism.

In this first case, they are not showing what some may describe as essential control data. When I first read that, it got my back up. Until I realised that the data isshown. Figures 1 and 2 both show bars where either no methylene blue has been added, and where no laser light has been added.

The menD SCV and its wild-type parent were also susceptible to photodynamic killing by methylene blue (20 μM) and 1.93 J/cm2 of 665 nm laser light, with reductions in cell viability of 3.5 log10 and 4.1 log10, respectively (data not shown)

Whilst we get a few details about what the menD data describes, the exclusion of this data is less justifiable.
The hemB SCV and its wild type paper when exposed to 20 μM methylene blue and 1.93 J/cm2 laser light have their cell viabilities reduced by ~2.5log10 and ~3.5 log10 respectively.

I should say that "Data not shown" is not quite the same as "Citation needed"- Reviewers can request to see the data, and then agree that it doesn't belong in the manuscript. The usual reason is that the data just isn't very good. The baseline controls may have been all over the place because someone left the incubator open too long, or some crucial piece of equipment broke down, and they could not repeat the experiment.
Accidents like these do crop up.
In this case, I strongly suspect the data was excluded because, as I have mentioned before, they were biasing their experiments against finding significance because they were using the wrong stats analysis. That is why they only present the general log10 reductions, but nothing about standard deviations and significance.
But that really isn't a justification for not showing the data. I shouldn't have to guess at what the data actually is, I should be allowed to read it first.

This paper does have a declared competing interest. This is not the first paper we have reviewed for #microtwjc to have a significant advertising push behind it *cough* manuka honey *cough*.
But the question is what effect such a competing interest woul have. In this case, the competing interest is thus :

Ondine Biopharma make the laser that was used in this study (It's branded Periowave). It is likely that the whole point of ST's studentship project was to investigate this technology, and if she couldn't find a use, we'd have to find her thesis to find out what the real story was. The fact that MW holds shares in Ondine could perhaps be problematic.
The question is how this could have affected the paper. There was some people talking about not being able to understand the methods, which is baffling because they were actually quite clear, requiring only some basic mathematics to fully understand. The only technology they are protecting is their laser, and frankly the audience they are pitching this article to wouldn't understand any of the patented technological details anyway.
So the "data not shown" could have been held back, not for the usual reasons of sheer embarrassment, but because some corporate paymaster decided it would hurt their shares if this menD data was published. Personally, I think authors already have a massive incentive to publish "positive" results that including a corporate entity merely allows the blame to be shifted if the research is eventually shown to be unethical.

In all, I stand by my first version of this review. It still basically does what it says on the tin. All it set out to do was show that an SCV can be killed by photodynamic therapy. It fits that narrow remit like a glove.

But you can't extrapolate to any treatment that could be useful, or even make general comments on multiple different SCV types, something that is quite important considering how the term "small colony variant" casts a vast umbrella over a massive variety of different bacterial mutations.
I was being slightly too nice to this paper, but because it set its target so low and managed to achieve it, I can't change my overall score for it. It is Okay.

*It can kill off mammalian cells. But we have a lot of those to lose, so we can survive Singlet Oxygen better.
** I presume this is the reason why these specific strains are described in terms of their growth rate rather than the specific antibiotic resistance they develop. But I haven't heard that theory since I was in school, so science could have moved on and I could be letting the tail wag the dog here. It could just as easily be that the growth rate of these bacteria slows because they are committing more energy to resisting antibiotics than growth.
***I would presume it would be a negative linear regression line if the treatment was 100% effective, and something like a reversed logistic regression line if high bacterial numbers diffused the effect of the light initially and there were some persistent survivors of the treatment. I may even be making things too complicated, and the data could fit to exponential decay.